PROJECT SUMMARY HIV persistence in the face of antiretroviral therapy necessitates lifelong treatment of infected individuals. HIV infection persists within a reservoir of CD4+ T cells that harbor latent integrated virus. The HIV reservoir may also reside in other long lived cell types such as macrophages, or may be maintained by HIV ongoing replication in the face of antiretroviral drugs (ARVs) due to lowered antiretroviral penetration in some anatomical compartments, or decreased sensitivity to ARVs due to cell-to-cell HIV spread. It is possible that multiple mechanisms of HIV persistence may play a role. Several approaches are being tested to target the HIV reservoir for elimination, including ?shock and kill? strategies, which reinitiate HIV gene expression and specifically eliminate infected cells following viral replication, and gene therapy approaches to excise the HIV genome. In addition, there are emerging strategies that recognize and kill infected cells directly. Absent from these approaches is a comprehensive understanding of the phenotypes and characteristics of the cellular source(s) of the HIV reservoir under antiretroviral therapy (ART). Lymph nodes may serve as a central reservoir for HIV persistence due to their high concentration of lymphocytes, extensive cellular interactions within the lymph node which may allow HIV spread between cells, and limited ARV penetration. Multiple CD4+ T cells subtypes have been proposed be the source of the HIV reservoir, but it is still unclear which cell subtype is most crucial to maintain it, and whether there is inter-patient heterogeneity in reservoir composition which depends on ART regimen or other clinical parameters. Furthermore, the composition of the HIV reservoir in Sub-Saharan Africa has rarely been investigated, despite the massively high prevalence of people living with HIV/AIDS in this region, as well as the extensive rollout of ART in at the epicenter of the HIV epidemic in South Africa and the consequent large and diverse ART suppressed patient population which can be studied to understand HIV persistence. In the proposed study, we aim to quantitatively determine the cell type(s) responsible for HIV production in the lymph nodes of ART-treated patients. We will use a novel approach utilizing single cell RNA-Seq to derive both the transcriptional profile of individual host cells and their intracellular viral transcript levels and viral sequences. This will enable us to deeply characterize which cell type(s) are infected, the number of viral transcripts per cell, and the transcriptional changes associated with infection. We will also determine the HIV reservoir size and composition in the lymph nodes of each ART suppressed individual by the established techniques of detecting HIV DNA copies and using viral outgrowth assays. We will test lymph nodes from a large number of study participants, enabled by a unique combination of very high infection prevalence and state of the art hospital facilities at the study's base in Durban. We will investigate common elements of the HIV reservoir across participants, and characterize reservoir heterogeneity.